The molecular mechanisms underlying narcotic tolerance and dependence remain poorly understood. Narcotic drugs primarily interact with the mu opioid receptor to produce acute effects, such as analgesia, and chronic effects, such as tolerance and dependence. A central goal of opioid research is to separate the acute from the undesirable chronic effects, in order to permit the safe use of narcotic analgesics. However, it remains to be shown whether the chronic effects result from the same signal transduction pathway via G proteins and second messengers, or from a distinct pathway. This project focusses on mu receptor regulation and cAMP signalling. We propose the hypothesis that upon agonist stimulation, the mu opioid receptor is slowly converted to a constitutively active state termed mu* (i.e., no longer requiring the presence of agonist) as a novel regulatory mechanism which had not been previously considered for neurotransmitter and hormone receptors. Further, constitutive activation to mu* is proposed to result from phosphorylation by a receptor kinase, a pathway which is independent of mu receptor coupling to second messengers. Since G protein coupled receptor (GPCR) kinases selectively phosphorylate activated receptors, the receptor can be trapped in the activated state mu* for prolonged time periods, thereby, driving narcotic tolerance and dependence. Initial results in SH-SYSY tissue culture, in U293 human kidney cells transfected with the mu receptor gene, and in an acute in vivo model of morphine tolerance and dependence in mice provide strong evidence in support of mu* playing a key role in tolerance and dependence. Kinase inhibitors were identified that prevent and reverse mu* formation in vitro and morphine tolerance and dependence in vivo, without affecting acute morphine effects. This proposal will focus on five main objectives. First, the biochemical regulation of mu receptor signal transduction via cAMP will be investigated in cells transfected with the mu receptor gene. Second, phosphorylation of the mu receptor will be studied in detail. Third, mu receptor kinases and phosphatases, and their regulation, will be studied as potential key factors determining narcotic tolerance and dependence. Fourth, kinase inhibitors will be identified as potential antiaddictive drugs. Fourth, all narcotic drugs will be reclassified according to their ability to affect C protein coupling and phosphorylation of mu and mu* for example, naloxone represents a mu antagonist with negative intrinsic activity, whereas the selective mu antagonist CTAP does not affect mu* activity (neutral antagonist) and was found to cause little withdrawal in morphine dependent mice. Neutral antagonists could serve in the treatment of narcotic dependence and overdose. The proposed studies will provide novel insights into narcotic addiction with broad potential therapeutic benefits.